Matrix metalloproteinases (MMPs) belong to the metzincin superfamily of zinc peptidases and include enzymes such as collagenase 3 (MMP 13), collagenase 1 (MMP 1), stromelysin 1 (MMP 3), gelatinase A (MMP 2), and gelatinase B (MMP 9) (Bode et al. (1996) Adv Exp Med Biol 389:1-11). MMPs are able to cleave components of the cartilage matrix. Accordingly, aberrant regulation of MMPs has been implicated in the collagen breakdown that occurs during certain diseases, such as rheumatoid arthritis and osteoarthritis (Shaw et al. (2000) Expert Opin. Investig. Drugs 9: 1469-78).
For instance, gelatinase levels have been shown to be significantly higher in the plasma of patients with rheumatoid arthritis (RA) and patients with RA complicated by amyloidosis or vasculitis as compared to healthy controls (Sopata et al. (1995) Rheumatol. Int. 15: 9-14). Expression of gelatinase B in fibrillated cartilage has been described as a useful marker of progressive articular cartilage degradation in osteoarthritis (Mohtai et al. (1993) J. Clin. Invest. 92: 179-85). Yoshihara and colleagues found increased levels of stromelysin-1 (MMP3) and a tissue inhibitor of metalloproteinases 1 (TIMP-1) in serum of rheumatoid arthritis patients as compared to osteoarthritis patients and healthy controls (Yoshihara et al. (1995) Arthritis Rheum. 38(7): 969-75; see also Garnero et al. (2002) Arthritis Rheum. 46(1): 21-30, and Walakovitz et al. (1992) Arthritis Rheum. 35(1): 35-42). In turn, plasma levels of MMP3 and TIMP1 have been shown to be significantly higher in osteoarthritis patients as compared to healthy subjects (Naito et al. (1999) Rheum. 38(6): 510-5). It has been suggested that MMP13 (collagenase 3) participates in tissue destruction in rheumatoid synovium and joint fluid (Lindy et al. (1997) Arthritis Rheum. 40: 1391-99), however the lack of an appropriate assay for MMP13 has been noted recently (Takei et al. (1999) J. Biomed. Mater. Res. 45: 175-83).
Once collagen is lost, it is rarely replaced. Therefore, the prevention of collagen degradation is an important aspect for the design of an effective treatment for rheumatoid arthritis and osteoarthritis, and research continues for effective inhibitors in view of this great unmet medical need (Elliott & Cawston (2001) Drugs Aging 18(2): 87-99). Early detection of the breakdown of collagen components would be a valuable first step in permitting the prevention of collagen degradation, by permitting the identification of patients in the early stages of arthritic disease.
In addition, MMPs are necessary for cancer progression, metastasis, and angiogenesis (new blood vessel formation). Because MMPs are able to degrade extracellular matrix (ECM) proteins, they are thought to facilitate tumor development by breaking down tissue barriers to invasion, thereby creating a path for tumor cells to colonize host tissues (Stamenkovic (2000) Semin. Cancer Biol. 10(6): 415-33). Numerous in vitro studies involving the use of inhibitors of MMP enzymes in reconstituted or cell-derived basement membrane systems support this view (Albini et al. (1991) J. Natl. Cancer Inst. 83: 775-79; DeClerk et al. (1991) Cancer Res. 51: 2151-57; DeClerk et al. (1992) Cancer Res. 52: 701-08; Khokha et al. (1992) J. Natl. Cancer Inst. 84: 1017-22). Recent evidence suggests that MMPs in the vicinity of tumors are produced by stromal cells rather than the tumor cells themselves, whereby the cancer cells induce stromal cells to synthesize MMPs using extracellular matrix metalloproteinase inducer (EMMPRIN) and cytokine stimulatory mechanisms in order to facilitate their invasion of tissues and local micrometastasis (Zucker et al. (2000) Oncogene 19(56): 6642-50). Thus, given that MMPs are required for the early steps of cancer progression and metastasis, detecting aberrant MMP activity can be an early indicator for the spread of invasive cancers.
For instance, Gohji and colleagues found that the mean serum level of MMP2 in prostate cancer patients was significantly higher in prostate cancer patients as compared to healthy controls and patients with benign prostatic hyperplasia (Gohji et al. (1998) Int. J. Cancer 78(3): 392-3). In another study, Hayasaka and colleagues showed that MMP9 levels were significantly higher in patients with hepatocellular carcinoma (HCC) as compared with healthy controls, patients with chronic hepatitis and patients with liver cirrhosis (Hayasaka et al. (1996) Hepatol. 24(5): 1058-62). Similarly, levels of MMP2 were found to be significantly elevated in the sera of Stage IV lung cancer patients as compared to normal sera (Garbisa et al. (1992) Cancer Res. 52(16): 4548-9; see also Hrabec et al. (2002) J. Cancer Res. Clin. Oncol. 128(4): 197-204). More recently, overexpression of stromelysin 3 (MMP11) was identified as a useful prognostic marker for the invasive potential of non-small cell lung cancer (Delebecq et al. (2000) Clin. Cancer Res. 6(3): 1086-92).
While elevated levels of particular MMP enzymes have been shown to be associated with particular types of cancer or imflammatory diseases, other groups have reported increases in particular MMPs over others in certain disorders. For instance, Riedel and colleagues reported a significant increase in MMP9 serum concentrations of patients with advanced stage head and neck squamous cell carcinomas as compared to patients with early stage cancer. However, no significant difference in MMP2 serum levels was observed (Riedel et al. (2000) Anticancer Res. 20(5A): 3045-9). In contrast, both MMP2 and MMP9 levels were found to be significantly higher in primary tumors from patients with either synchronous or metachronous metastases as compared to patients who were disease-free following radical nephrectomy (Slaton et al. (2001) Am. J. Pathol. 158(2): 735-43). Thus, the level of particular MMPs may vary depending on the type and/or stage of cancer, therefore specific diagnostics tests that are capable of distinguishing between various MMPs would be valuable in diagnosing different types of cancer. Furthermore, given the lack of sensitivity and specificity of substrates used in the prior assays, more sensitive substrates would permit an accurate evaluation of which MMPs show elevated expression in particular patient populations.
Assays to measure (MMP) activity in biological fluids have been described elsewhere (Verheijen et al. Biochem J. (1997) May 1; 323(Pt 3): 603-9; Beekman et al. (1999) Ann NY Acad Sci. June 30; 878: 150-8; Beekman et al. (1997) FEBS Lett. December 1; 418(3): 305-9; Beekman et al. (1996) FEBS Lett 390: 221-5) and commercially available kits exist (Amersham Biotrak™ ELISA system, RPN2632), but none are specific or sensitive enough or designed to measure MMP activity directly in bodily fluids and thus, are not of clinical benefit to the patient. Obtaining a profile of MMP activity is important because it may serve as a predictor of disease progression or even a diagnostic tool in diseases such as osteoarthritis (OA), rheumatoid arthritis (RA) and cancer. Thus, research to find informative markers that can serve as dosimeters of these diseases continues, as there are currently no robust and reliable commercial tests.
For instance, a colorimetric assay was developed (Verheijen et al., 1997) that relies on cleavage of a sequence preferred by MMPs within a full length protein. This colorimetric procedure was used for a MMP-9 assay that measures gelatinase B activity in urine (Hanemaaijer et al. (1999) Ann. N.Y. Acad. Sci. 878: 141-9). This technique may not, however, be sensitive enough to measure collagenase 3 levels in biological fluids where picomolar amounts are present.
Preliminary data with fluorescent peptide substrates to measure MMP activity in synovial fluid have been reported and are sensitive, but the peptides did not have a proper sequence so as to confer enough selectivity to permit an accurate measurement of collagenase 3 activity in biological samples such as synovial fluid, plasma and urine. MMPs such as the gelatinases, collagenase 1 and stromelysin are present in abundant quantities, but the currently available collagenase 3 substrate is only 8, 3, and 60 fold selective over gelatinase B, gelatinase A, and stromelysin (Beekman et al, 1999), respectively. Unfortunately, stromelysin is an enzyme that is prevalent in synovial fluid and blood plasma during disease states such as rheumatoid arthritis and is therefore likely to contribute to the measurable activity when non-specific substrates are employed (Yoshihara et al. (1995)).
Kits are commercially available from Amersham that measure MMP activities using a calorimetric assay in a 96-well plate format. Semi-specific antibodies are used to pull a given MMP out of the biological fluid and then activity is measured via addition of a substrate that produces a color upon cleavage. Several problems exist with these kits. First, since the antibodies are not completely specific, a single enzyme assay may in fact measure a composite of MMP activities. Second, the assay is colorimetric and is not very sensitive. For instance, the Amersham kit is only capable of detecting nanomolar levels of MMP enzyme, as compared to the novel substrates disclosed herein, which detect picomolar quantities of collagenase 3 activity. Finally, for at least the Amerhsam collagenase 3 kit, the signal detected is independent of antibody addition, suggesting that the kit does not in fact accurately measure collagenase 3 levels.
Another way in which to assess MMP activity is with neo-epitope antibodies to the cleavage products of substrates. This technique has been used to assess disease activity in patients with rheumatoid and osteoarthritis, where efforts have focused on the proteases that degrade type II collagen, the principle collagen of the joint. Poole and coworkers discovered that the major cleavage point of type II collagen by the matrix metalloproteases 1 and 13 (collagenase 1 and 3) occurs at the sequence GPQGLAGQ (SEQ ID NO: 1). Using neo-epitope Abs to that sequence, they detected processed collagen fragments in synovial fluid from rheumatoid or osteoarthritic patients (Billinghurst et al. (1997) J. Clin. Invest. 99: 1534-1545; Dahlberg et al. (2000) Arthritis Rheum. March; 43(3): 673-82). These neoepitope antibodies are not commercially available. Moreover, the sequence detected by this antibody, GPQG-NH2, can arise from cleavages by other MMP enzymes.
Another neo-epitope antibody to the sequence at the 3/4 collagen I or II cleavage site has been created by Pfizer (Huebner et al. (1999) Trans. Orthop. Res. Soc. 25: 198). This antibody, 9A4, recognizes the sequence GPP(OH)GPQG—COOH (SEQ ID NO: 2) (Huebner et al. (1998) Arthritis Rheum. 41: 877-890). Together with an upstream anti-collagen II specific antibody, collagen fragments are detected in urine (the urinary type II collagen neo-epitope assay or uTIINE) (Saltarelli et al. (1999) Arthritis Rheum. 42(9): 1071). The urinary TIINE activity has been shown to correlate with OA disease activity (Woodworth et al. (1999) Arthritis Rheum. 42(9): 1125), thereby demonstrating the importance of collagenase activity in the osteoarthritis disease process. These assays, however, are not commercially available and do not discern the particular collagenase or relative contribution of the various collagenases in different arthritides.
Recently, a role for collagenase 3, gelatinase A and gelatinase B in the processing of protein substrates has been discovered by utilizing substrate mapping with phage display (Deng et al. (2000) J. Biol. Chem. 275: 31422-7; Kridel et al. (2001) J Biol Chem. 276(23): 20572-8; Chen et al., J Biol Chem (IN PRESS)). Substrate mapping with phage display has also been performed with stromelysin 1, although the information was not used to design very specific substrates, nor were physiological substrates found for this enzyme (Smith et al. (1995) J Biol Chem. March 24; 270(12): 6440-9). In the case of collagenase 3, peptide substrates are cleaved that display sequence homology with type IV collagen, biglycan, and TGF-beta-3 (Deng et al, 2000).
Using the previous phage display data as a starting point, the present inventors were successful in obtaining many substrate sequences showing enhanced selectivity for collagenase 3 over the other matrix metalloproteases. In the process of identifying these substrates, the inventors identified several structure/function relationships useful for the design of substrates having specificity and selectivity for collagenase 3. Further, in the process of designing assays for the use of these substrates, the present inventors identified several improvements over existing assays that would increase the selectivity and sensitivity of MMP assays in general. These novel substrates for collagenase 3 and improved MMP assays are useful tools for measuring MMP activity in disease states where levels/activities are discordant in comparison with healthy subjects.